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Membrane-spanning peptides induce phospholipid flop: a model for phospholipid translocation across the inner membrane
M A Kol1, A I de Kroon, D T Rijkers
1Department Biochemistry of Membranes, Center for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. m.a.kol@chem.uu.nl
Abstract:
The mechanism by which phospholipids translocate (flop) across the E. coli inner membrane remains to be elucidated. We tested the hypothesis that the membrane-spanning domains of proteins catalyze phospholipid flop by their mere presence in the membrane. As a model, peptides mimicking the transmembrane stretches of proteins, with the amino acid sequence GXXL(AL)(n)XXA (with X = K, H, or W and n = 8 or 12), were incorporated in large unilamellar vesicles composed of E. coli phospholipids. Phospholipid flop was measured by assaying the increase in accessibility to dithionite of a 2,6-(7-nitro-2,1,3-benzoxadiazol-4-yl)aminocaproyl (C(6)NBD)-labeled phospholipid analogue, initially exclusively present in the inner leaflet of the vesicle membrane. Fast flop of C(6)NBD-phosphatidylglycerol (C(6)NBD-PG) was observed in vesicles in which GKKL(AL)(12)KKA was incorporated, with the apparent first-order flop rate constant (K(flop)) linearly increasing with peptide:phospholipid molar ratios, reaching a translocation half-time of approximately 10 min at a 1:250 peptide:phospholipid molar ratio at 25 degrees C. The peptides of the series GXXL(AL)(8)XXA also induced flop of C(6)NBD-PG, supporting the hypothesis that transmembrane parts of proteins mediate phospholipid translocation. In this series, K(flop) decreased in the order X = K > H > W, indicating that peptide-lipid interactions in the interfacial region of the membrane modulate the efficiency of a peptide to cause flop. For the peptides tested, flop of C(6)NBD-phosphatidylethanolamine (C(6)NBD-PE) was substantially slower than that of C(6)NBD-PG. In vesicles without peptide, flop was negligible both for C(6)NBD-PG and for C(6)NBD-PE. A model for peptide-induced flop is proposed, which takes into account the observed peptide and lipid specificity.
Insights
Transmembrane protein domains can catalyze phospholipid movement across bacterial membranes. Mimicking peptides accelerated phospholipid translocation, supporting this mechanism and revealing lipid-specific interactions.
Area of Science:
- Biochemistry
- Membrane Biology
- Molecular Biophysics
Background:
- Phospholipid translocation (flop) across the E. coli inner membrane is not fully understood.
- The role of transmembrane protein domains in catalyzing phospholipid flop requires investigation.
Purpose of the Study:
- To test the hypothesis that membrane-spanning protein domains can induce phospholipid flop.
- To investigate the influence of peptide sequence and lipid type on the rate of phospholipid translocation.
Main Methods:
- Incorporation of synthetic peptides mimicking transmembrane protein segments into large unilamellar vesicles.
- Measurement of phospholipid flop using a dithionite accessibility assay with a fluorescently labeled phospholipid analogue (C(6)NBD-PG and C(6)NBD-PE).
Main Results:
- A peptide (GKKL(AL)(12)KKA) significantly accelerated C(6)NBD-phosphatidylglycerol (PG) flop, with rate increasing linearly with peptide concentration.
- Other tested peptides also induced PG flop, supporting the role of transmembrane segments.
- Flop rates were dependent on amino acid identity (K > H > W) and phospholipid type (PG faster than PE).
Conclusions:
- Transmembrane protein domains can indeed catalyze phospholipid translocation across membranes.
- Peptide-lipid interactions at the membrane interface modulate the efficiency of flop.
- A model for peptide-induced phospholipid flop, considering peptide and lipid specificity, is proposed.